Although a diverse arsenal of antimicrobial drugs has been developed, bacterial and fungal infections remain prevalent worldwide. The formation of multicellular communities of microbes, called biofilms, is a common feature of these diseases that contributes to their endurance. Cells in biofilms are held together by an extracellular matrix and differ from those grown in liquid cultures with respect to their metabolism and sensitivity to antimicrobial drugs. Our long-term goal is to define the integrated, redox-based regulatory networks and metabolisms that support survival of bacteria in biofilms, with a focus on the pathogen Pseudomonas aeruginosa. We hypothesize that the changing redox states of cells in developing biofilms affect multicellular behavior, virulence, and susceptibility to antibiotics. A standardized colony biofilm morphology assay facilitates our investigation of mechanisms that underpin biofilm-specific metabolism and feature formation. We will use basic molecular approaches combined with newly developed and innovative techniques, including electrochemical and microscopic analysis of biofilms at the micron scale, to address fundamental questions regarding the use of redox-active substrates for metabolism and the global regulation of biofilm matrix in response to redox cues: (1) How does P. aeruginosa balance its cellular redox state in the oxygen- limited regions of biofilms? (2) How does redox control of cellular signaling determine overall community morphology and cellular arrangement within the biofilm? (3) How does the use of pyruvate and lactate, important energy sources and metabolic intermediates, contribute to survival in biofilms and metabolism during host infection? As P. aeruginosa is a major cause of infections in hospitals and in patients with cystic fibrosis, we are motivated by the potential for these mechanisms to serve as therapeutic targets.